A vaccine capable of targeting and neutralizing sporozoites, the infectious stage of the malaria parasites injected by mosquito bite, remains a promising target for preventing malaria infection and transmission. Analysis of malaria vaccine trials has shown that antibodies can neutralize the sporozoite and prevent infection of the liver. However, the most clinically advanced pre-erythrocytic vaccine candidate based on the circumsporozoite protein (CSP) confers only partial protection, indicating that it is necessary to add new antigen targets to increase vaccine efficacy. The overall goal of this proposal is to evaluate novel Plasmodium falciparum pre-erythrocytic antigen candidates that can elicit protective immunity from infection. Building on our recent unprecedented characterization of the P. falciparum sporozoite surface/secreted proteome, we have identified a number of promising surface/secreted antigens that warrant interrogation as new vaccine candidates. Based on this evidence, we will develop 15 P. falciparum sporozoite surface/secreted antigens in a state-of-the-art recombinant protein expression platform and evaluate whether they can elicit protective immunity against mosquito bite sporozoite infection. We will employ an innovative combination of transgenic Plasmodium parasite technology, B cell cloning techniques and humanized rodent models to develop a comprehensive pre-erythrocytic antigen development pipeline for analysis of antibody-mediated protection. In addition, we will evaluate the mechanisms of vaccine-elicited protective humoral immunity against the parasite and deepen our understanding of B cell-mediated protective immunity. Our experimental systems to study the human pathogen P. falciparum in a fully humanized humoral immunogenicity mouse model and a liver- humanized mouse challenge model will ensure our results provide the closest possible approximation of vaccination-elicited responses in humans. The outcomes of our aims will provide an evidence-based selection of novel vaccine candidates and will accelerate their pathway toward human clinical trials using the P. falciparum challenge model. If successful, novel non-CSP vaccine immunogens identified can be considered for inclusion into next generation multi-antigen subunit malaria vaccines.